Jae Min Cho^1,2†, Seul-Ki Park^1,2†, Oh Sung Kwon^3,4†, David Taylor La Salle^1,5, James Cerbie^1,5, Caitlin C. Fermoyle^1,5, David Morgan⁶, Ashley Nelson⁵, Amber Bledsoe⁶, Leena P. Bharath^1,2, Megan Tandar¹, Satya P. Kunapuli⁷, Russell S. Richardson^1,5,8, Pon Velayutham Anandh Babu¹, Sohom Mookherjee^1,2, Bellamkonda K. Kishore^1,9,10, Fei Wang^9,10, Tianxin Yang^9,10, Sihem Boudina^1,2, Joel D. Trinity^1,5,8, and John David Symons^1,2*

¹Department of Nutrition and Integrative Physiology, University of Utah, Salt Lake City, UT, USA; ²Division of Endocrinology, Metabolism and Diabetes, Program in Molecular Medicine University of Utah School of Medicine, Salt Lake City, UT, USA; ³Department of Kinesiology, University of Connecticut, Storrs, CT, USA; ⁴Department of Orthopedic Surgery & Center on Aging, University of Connecticut School of Medicine, Storrs, CT, USA; ⁵Geriatric Research, Education, and Clinical Center, George E. Whalen Veterans Affairs Medical Center, Salt Lake City, UT, USA; ⁶Department of Anesthesiology, University of Utah, Salt Lake City, UT, USA; ⁷Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA; ⁸Department of Internal Medicine, Division of Geriatrics, University of Utah, Salt Lake City, UT, USA; ⁹Nephrology Research, George E. Whalen VA Medical Center, Salt Lake City, UT, USA; and ¹⁰Department of Internal Medicine, Division of Nephrology, University of Utah, Salt Lake City, UT, USA

^* Corresponding author.
^† These authors contributed equally.

Aim
The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier, we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signalling to endothelial NO synthase (eNOS). Here, we illustrate the translational and functional significance of these findings.

Methods and results
First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperaemia evoked by rhythmic handgrip exercise-elevated radial artery shear-rate similarly from baseline in adult and older subjects for 60 min. Compared with baseline, indexes of autophagy initiation, p-eNOS^S1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOS^S1177 activation were robust in ECs from adult but not older animals that completed 60-min treadmill-running. Furthermore, 20 dyne • cm² laminar shear stress × 45-min increased autophagic flux, glycolysis, ATP production, and p-eNOS^S1177 in primary arterial ECs obtained from adult but not older mice. Concerning functional relevance, we next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOS^S1177in vitro precipitates arterial dysfunction ex vivo. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) purinergic 2Y₁-receptor (P2Y₁-R) blockade; and (v) germline depletion of P2Y₁-Rs. Importantly, P2Y₁-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy.

Conclusions
Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y₁-Rs.